Contamination of water with hazardous wastes such as toxic organic chemicals, metals or radionuclides is a problem of ever-increasing urgency. In particular, groundwater aquifers at many industrial and defense-related sites are contaminated with one- and two-carbon, volatile halogenated aliphatic hydrocarbons such as trichloroethylene ("TCE"), many of which are suspected carcinogens. (See, e.g., J. J. Westrick et al., J. Am. Water Work Assoc. 5:52 (1984) and P. F. Imfante and T. A. Tsongas, Environ. Sci. Res. 25:301 (1982)). Due to the natural flow of groundwater and dispersive transport processes, aquifer contamination at such sites has usually resulted in large, dilute migrating plumes that can extend tens of meters in depth. The contaminants are present in the aqueous phase and they are sorbed on the mineral phases that constitute a heterogeneous soil or rock. These physical characteristics of aquifer contamination make groundwater remediation to the concentrations mandated by the EPA (less than or equal to 5 ppb for TCE) a challenging problems.
Recent legislation regulating the disposal and cleanup of such hazardous wastes has led to an ongoing search for new treatment techniques. However, many of the now-developed technologies are not cost-effective, nor are they easily implemented. For example, current technology for remediating volatile organic compounds is essentially limited to "pump-and-treat" methods. In such methods, as described, for example, by H. H. Russell et al. in Remediation 1:167 (1990), the plume is penetrated by a number of wells, the contaminated ground water is extracted from the wells and pumped to the surface, treated by one of a number of methods at the surface, and then either injected into the subsurface or discharged. The pump-and-treat approach is expensive to operate, however, and there is significant uncertainty in the ultimate level of decontamination, the time required to achieve significant decontamination, and the permanence of decontamination. See, e.g., C. C. Travis et al., Environ. Sci. Technol. 24:1464 (1990), and K. H. Baker et al., Geomicrobiol. 8:133 (1991). The main cause of these uncertainties is the highly heterogeneous nature of the subsurface medium, which creates preferential flow paths for the extracted fluids. Less permeable subsurface regions receive less remediation and remain as sources of residual contamination to recontaminate the cleaned up regions. Additionally, many methods other than the pump-and-treat technique cannot permanently clean up water contaminated with certain types of hazardous materials.
In situ microbial bioremediation of aquifers contaminated with volatile organic compounds has received increasing attention as an alternative to the pump-and-treat technique. It has the potential advantages that many common organic contaminants can be biodegraded to innocuous compounds by naturally occurring microorganisms, that it is a contaminant-destructive as opposed to a contaminant-relocative process, and that it is carried out in situ, obviating the need for disposal of the treated groundwater. Unfortunately, clear-cut proof that biodegradation of the undesired organic contaminant has taken place in an aquifer has been difficult to substantiate, even for TCE added in very small amounts to a very small, slow aquifer.
The usual approach to in situ bioremediation is to pump a suite of nutrients into the subsurface to stimulate the growth of indigenous bacterial populations. Such methods are described, for example, in the following references: K. H. Baker et al., Geomicrobiol. 8:133 (1991); J. T. Wilson et al., Ground Water Monit. Rev., Fall 1986, at page 56; M. D. Lee et al., CRC Crit. Rev. Environ. Control 18:29 (1988); P. V. Roberts et al., Ground Water 28:591 (1990); L. Semprini et al., Ground Water 28:715 (1990); L. Semprini et al., Ground Water 29:239 (1991); L. Semprini et al., Ground Water 29:365 (1991); P. E. Flathman, Ground Water Monit. Rev. 9:105 (1989); and J. T. Wilson et al., Appl. Environ. Microbiol. 49:242 (1985). Ideally, the resulting increase in biomass causes the desired biodegradation at an acceptable rate. However, three complications arise: (1) heterogeneous permeability of the subsurface environment makes it difficult to deliver nutrients through the contaminated plume; (2) nutrient pumping often causes preferential growth near the injection wells and can lead to biofouling; and (3) the biotransformation of halogenated hydrocarbons is generally a cometabolic phenomenon.
In a recent field study, in situ methanotrophic bioremediation was evaluated in a shallow, semiconfined aquifer that was about 1.5 m thick and consisted of fine- to coarse-grained sands and gravel (P. V. Roberts et al., Ground Water, 28:591 (1990); L. Semprini et al., Ground Water, 28:715 (1990); L. Semprini et al., Ground Water, 29:239 (1991); L. Semprini et al., Ground Water, 29:365 (1991)). Dissolved methane and oxygen were injected into the aquifer in alternating pulses to stimulate the growth of indigenous methanotrophs. Monitoring wells were located at 1, 2.2 and 3.8 m and an extraction well was situated 6 m downstream from the injection well. After several weeks of gas injection, biostimulation of the aquifer was achieved, but the methane-oxidizing bacteria grew preferentially within 2 m of the injection well. When TCE (97 ppb and later 51 ppb) was then injected, along with more methane and oxygen, the maximum biodegradation attained within 2 m of the downstream travel was only 20- 30%. This field study suggests that the periodic pumping of a limited supply of methane plus air as a method to replenish a spent biofilter, promotes localized methanotrophic bacterial growth. The more obvious replenishment method would be simply to inject additional bioreactor grown cells.
The present invention is directed to the aforementioned problem, i.e., the removal of potentially hazardous contaminants from fluids, and involves the use of bioremediation. Specifically, the invention is premised on the discovery that certain microorganisms capable of producing an enzyme which degrades one or more targeted contaminants may be grown in such a way as to maximize the production and intracellular stability of that enzyme or enzyme systems, and further that such catalytically active, intact microorganisms may be injected directly into a pool or flow of contaminated fluid to remove contaminants therefrom. The invention is particularly useful in eliminating a number of toxic halogenated aliphatic and various aromatic organic chemicals from groundwater and the approach can be applied to other types of contaminants such as metals or radionuclides. The invention may be used to treat a contaminant source as well as a flowing contaminated stream. The bioremediation process of the present invention may also be used to treat small volumes of large, moving plumes or may be translated into a larger-scale context.